Approximately 750,000 total hip and knee replacement surgeries are performed each year in the United States. As a result of the aging population, these numbers are expected to increase to two and a half million by 2030. While the majority of those who undergo these procedures experience dramatic benefit, approximately three percent require re-operation, most commonly for prosthetic joint infection (PJI) or aseptic loosening of the prosthesis. Surgical and medical management of failed joint replacements depend on whether or not infection is present. However, making a microbiologic diagnosis of PJI is challenging. We have shown that native joint infection diagnostics are inaccurate for PJI. Microorganisms associated with PJI are found in biofilms on the prosthesis surface; accordingly, methods that sample the prosthesis surface should improve the diagnosis of PJI. Our research team published a manuscript in the New England Journal of Medicine, showing that a novel technique that that we have developed, which samples biofilms on the prosthesis surface, is more sensitive than conventional periprosthetic tissue for the culture-based diagnosis of PJI. Although this technique is more sensitive than conventional approaches, it is culture-based, and, accordingly has a long turnaround time. Further, despite improved sensitivity compared to periprosthetic tissue culture (i.e., 78.5 versus 60.8%, p < 0.001), there remain culture negative cases. We hypothesize that a molecular microbiologic approach, targeting biofilm bacteria dislodged from explanted orthopaedic prostheses, will more sensitively, and rapidly, detect PJI compared with our culture-based approach. We further hypothesize that bacteria in biofilms, identified using molecular methods, are associated with some cases of 'aseptic' failure of prosthetic joints, the pathogenesis of which is incompletely defined. We are developing and validating closed system rapid real-time polymerase chain reaction assays, collectively targeting bacteria which cause PJI, for detection of biofilm bacteria dislodged from the surface of orthopaedic prostheses. We will prospectively evaluate these assays to confirm improved sensitivity compared to our culture- based approach, for the diagnosis of PJI. We will determine the percentage of revision hip and knee arthroplasties performed for reasons other than PJI that have previously unrecognized bacteria present at revision, as detected by molecular techniques that detect bacterial biofilms, and we will assess the outcome of such cases. Our research team has extensive translational experience in the area of PJI and has collaborated on a number of studies. Collectively, we have expertise in molecular microbiology, bacteriology, orthopaedic infection and surgery, medical biofilms, histopathology, and biostatistics. Results of this study are expected to yield a novel, rapid, and sensitive molecular diagnostic for PJI, and will assess whether microbes are associated with aseptic hip and knee arthroplasty failure, both of which will transform clinical practice.